Our long-term goals are to understand the spatial and temporal events involved in gene expression/silencing in living cells. Understanding the dynamics of these events in vivo will provide the basis to identify changes that occur in cells or tissues associated with various disease states. We propose to utilize a live cell system to allow us to visualize a fundamental cellular process, gene silencing, at the levels of DNA, RNA, and protein, directly in living cells. This system will be used to assess the assembly and dynamics of Polycomb group proteins at a regulatable genetic locus. Using this system we will characterize the temporal pattern of factor arrival and release, and correlate it to changes in chromatin structure. In addition, we will study the dynamics of this specific genetic locus during the transition from the "on" to the "off" transcriptional state, in relation to RNA clearance and nuclear organization. The development and utilization of this live cell system will allow us to gain important insight into the spatial and temporal aspects of gene silencing that cannot be obtained by previously used approaches. [unreadable] [unreadable]